Recognition of the complexities of inositol lipid signaling has increased dramatically in the last several years. PtdIns(4,5)P2 directly binds and regulates activity of many proteins, and at least three major second messengers are produced in a single step from this minor membrane phospholipid. The inositol lipid-specific phospholipase C isozymes integrate signals from multiple upstream G protein regulators, and act as highly organized but poorly understood signaling nodes central to the action of many growth factors, hormones, neurotransmitters, and other stimuli. Recent work from our laboratories has led to increased appreciation of multiple modes of regulation of inositol lipid signaling by Rac, Rho, and heterotrimeric G proteins. Moreover, our structures of PLC-22 and of PLC-22 in GTP-dependent complex with Rac1 provide the first mechanistic snapshots of PLC isozymes past the original structure of PLC-4 solved a decade ago. This progress and recent success in producing stable complexes of G1q and PLC-2 isozymes has placed us in position to design experiments that will provide understanding of G protein-mediated regulation of the catalytic activity of PLC isozymes in structurally and mechanistically unambiguous terms. We propose three specific aims to reach this goal: Specific Aim 1. To delineate the interface and mechanism through which GTP-bound Rho binds PLC-5 and cooperates with Ras in activation of this novel PLC isozyme. Specific Aim 2. To define the structural basis of interaction of G1q with the carboxy-terminus of PLC-2 isozymes, and to delineate the mechanism through which this interaction stimulates PLC activity. Specific Aim 3. To determine the mechanism whereby an auto-inhibiting occlusion of the active site by a catalytic domain insert is relieved during interfacial activation of PLC isozymes at phosphoinositide-containing membranes.